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Description  |
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FIELD OF THE INVENTION
The present invention relates, in general, to compositions, methods and
diagnostic kits useful for detecting microorganisms associated with
vaginal disorders. In one aspect, the invention relates to methods for
releasing intact nucleic acid from a microorganism. In another aspect, the
invention relates to compositions of oligonucleotide probes for use in the
detection of microorganisms associated with vaginal disorders. Methods for
detection as well as diagnostic kits for the assay of these microorganisms
are also disclosed.
BACKGROUND OF THE INVENTION
One of the most common reasons women seek medical treatment is for vaginal
discharge or other vaginal symptoms. In women who visit their physician
with vaginal complaint, approximately 40% are diagnosed as having some
form of vaginitis, and 90% of these cases fall into three clinical
entities: bacterial vaginosis (BV), trichomoniasis, and vulvovaginal
candidiasis. (See, e.g., Sobel, "Vaginal Infections in Adult Women,"
Medical Clinics of North America 74:1573 (1990)). The symptoms of these
three distinct diseases overlap, thus creating a need for differential
diagnosis before appropriate and specific medication can be prescribed. A
rapid and accurate diagnosis is especially critical in pregnant women, in
whom BV and trichomoniasis are associated with premature births and low
birth weight babies. Moreover, BV-positive pregnant women are predisposed
to chorioamnionitis, amniotic fluid infection, and puerperal infectious
morbidity. BV has also been associated with pelvic inflammatory disease,
postpartum endometritis, bacteremia, salpingitis, and the like.
The term "bacterial vaginosis" was coined only a few years ago, the disease
being formerly known as "leukorrhea" or "non-specific" vaginitis. Until
the past decade, the cause of this syndrome was presumed to be some
unidentified pathogen. A study published in 1955 suggested that
Gardnerella vaginalis was the causative agent of BV, but this proposition
was discredited by subsequent studies revealing that G. vaginalis was
present in the vaginal secretions of 10-50% of normal women, i.e.,
BV-negative women. Since then it has become apparent that, unlike most
diseases, BV cannot be attributed to one specific etiologic agent, but
instead results from a drastic alteration of the vaginal flora. The
normally present Lactobacilli become greatly reduced in number, and there
is a concomitant overgrowth of several anaerobic bacteria and other
microorganisms, especially Gardnerella vaginalis (Gv). This alteration is
accompanied by an increase in vaginal pH.
The clinical "gold standard" method of diagnosing BV involves the
examination of four criteria, and does not involve microbiological
culture:
1) presence of clue cells (determined microscopically);
2) white or gray adherent homogeneous discharge;
3) vaginal fluid pH>4.5; and
4) fishy amine odor when vaginal fluid is mixed with 10% potassium
hydroxide (KOH).
To diagnose BV, some investigators require the presence of clue cells plus
two of the other three indicators, while other investigators require only
that any three of the four indicators be present. In practice, physicians
do not typically conduct pH and amine odor tests in their offices, nor
even attempt to identify clue cells. In fact, use of the gold standard
test is confined primarily to clinical studies. Identification of clue
cells requires special skills, since such cells are difficult to
distinguish from other microscopically observable entities. Clue cells are
not microorganisms, but are vaginal epithelial cells that have been shed
from the vaginal wall and to which a large number of rod-shaped bacteria
have adhered. The adherent cells include G. vaginalis, and other anaerobic
species including, for example, Mobiluncus species.
Another consistent hallmark of BV is the elevation of vaginal pH above the
normal value of 4.5. Unfortunately, this highly sensitive indicator lacks
specificity, as conditions other than BV can also cause an elevated
vaginal pH. For example, infection with Trichomonas vaginalis or
cervicitis can cause the vaginal pH to go up. Hence, vaginal pH by itself
cannot be used to diagnose BV because such a practice would result in an
unacceptable incidence of false positives.
In addition to the gold standard criteria, BV is sometimes diagnosed by
assessing the shift in vaginal flora by examining Gram stained vaginal
smears. This method, used primarily in research protocols, is difficult to
perform and requires special training, thereby rendering it unsuitable for
physician's offices. Moreover, this technique is less sensitive and less
specific for BV than the gold standard method. (See, e.g., Nugent, et al.,
"Reliability of Diagnosing Bacterial Vaginosis Is Improved By A
Standardized Method of Gram Stain," J. Clin. Microbiol. 29(2):297-301
(1991).
Currently, some physicians make use of a wet mount in conjunction with
office vaginal examinations. A slide prepared from the patient's vaginal
fluid is visually examined by the physician. When a BV-positive patient is
examined by a physician practiced in making these difficult observations,
such a slide will reveal an absence of the usual levels of Lactobacilli,
which are large rods, and the presence of a large number of small
rod-shaped bacteria, including Gardnerella vaginalis (Gv), Prevotella, and
Mobiluncus species. The former two bacteria have straight rod shapes,
while the latter bacterium exhibits a curved rod shape. Some physicians
believe that clue cells may be identified through wet mount analysis, but
such means of identification are not generally accepted as appropriate.
When fast isolated, G. vaginalis was termed Haemophilus vaginalis. Later,
G. vaginalis was reclassified as Corynebacterium vaginalis. Finally, G.
vaginalis was placed into a new genus, Gardnerella, as it did not properly
belong in either of the first two classifications. As such, some
investigators have attempted to determine whether the amount of G.
vaginalis present in a sample is indicative of BV. In doing so, they
concluded that BV-positive women, on the average, have higher levels of G.
vaginalis than BV-negative women. Considerable overlap was found to exist
in the levels of G. vaginalis found in BV-positive and BV-negative women,
however, thereby rendering the G. vaginalis cell level inconclusive
evidence of the disease state. See, Amsel, et al., Am. J. Med. 74:14-22,
1983 and Eschenbach, et al., Am. J. Obstet. Gynecol. 158:819-28, 1988.
BV is one common cause of vaginal complaints. Other microorganisms commonly
associated with such symptoms are Candida species and Trichomonas
vaginalis. The most typical way of diagnosing candidiasis is according to
symptoms, visual inspection of the vagina, and microscopic detection of
the organism itself. For the wet mount, potassium hydroxide is added to
dissolve epithelial cells, and the slide is examined for the presence of
yeast elements, for example, pseudohyphae or budding yeast. If these
measures do not yield a definitive diagnosis, the physician may order a
culture. An alternative to culture method is Gram stain, which requires a
trained person to analyze the results.
The classical method for the diagnosis of Trichomonas involves
demonstration that the organism is present. Trichomonas is not a normal
inhabitant of the vagina, and is considered a pathogen anytime it is
detected. Typically, detection is done microscopically by observing
protozoa with characteristic motility in vaginal secretions mixed with
saline in a wet mount. Since Candida wet mounts contain potassium
hydroxide, separate wet mounts must be used if one wishes to look for both
of these organisms. Detection of Trichomonas depends on observation of
flagellated cells of a characteristic size and shape that are in motion.
Unfortunately, trichomonads quickly lose their distinctive motility upon
cooling to room temperature, therefore, a microscope and trained
microscopist must be available immediately after the sample is taken. Once
they have lost their motility, trichomonads are practically
indistinguishable from lymphocytes present on the slide. To exacerbate the
challenge of microscopically detecting trichomonads is the fact that they
tend to be present in low numbers.
In view of the foregoing, it is readily apparent that there are numerous
disadvantages associated with the use of culture for diagnosing vaginal
disorders, particularly if the woman presents with symptoms of vaginitis.
The foremost disadvantage is the three to seven days required to obtain
culture results. This delay can lead doctors to avoid culture altogether
and, instead, to dispense medication based on a less accurate method of
microscopic examination of a wet mount.
Moreover, aside from the delay in getting the results, culture can be
prohibitively expensive when the syndrome can be caused by three different
etiologic agents, as is the case with vaginitis. Even if a patient were
willing to pay, most commercial microbiology laboratories do not offer
Trichomonas vaginalis culture. Moreover, even when this culture is
available, logistical problems arise from trying to culture three
organisms from a single patient. If one swab is used and placed into the
standard bacterial transport medium, the Trichomonas will not survive.
This fastidious organism requires a specialized transport medium. Hence,
at least two swabs must be taken. In fact, the microbiologist would prefer
a separate swab for each organism to be cultured. Yet if three swabs are
taken, it is not likely that all three will pick up identical samples, as
the successive swabs are likely to deplete the vaginal fluid, and may even
cause irritation.
In the case of Gardnerella vaginalis and Candida albicans, culture is of
limited utility because these organisms can be present in the non-diseased
vagina. In many instances, culture for these organisms would have
diagnostic value if it were designed to yield quantitative data that could
be used to identify clinically significant levels of these organisms, a
procedure that involves plating serial dilutions of each sample. But,
routine culture protocols do not involve plating serial dilutions to
identify clinically significant levels and, thus, they determine only
whether the organism is present. At best, the microbiology laboratory will
inform the physician whether the growth was heavy or light. This limited
information is not sufficient for the diagnosis of BV or candidiasis.
Even if a method were available for analyzing a single swab for the
presence of multiple organisms, there are numerous drawbacks of culture
and wet mount. As such, a biochemical test would be more economical than
culturing for several different organisms. Moreover, if the test could be
performed in less than an hour, the diagnosis could be completed before
the patient left the doctor's office, thus enabling her to obtain the
correct medication that same day.
One advantage of culture is that the organism is given a chance to multiply
before being identified. However, since a swab can pick up only limited
amounts of sample, a successful biochemical method would have to possess
the capability of detecting very small numbers of organisms. As such, a
biochemical method performed in the doctor's office would have to be able
to yield results from the minuscule amount of sample present on one or two
swabs. For tests that rely on detecting cytoplasmic components of the
pathogenic organisms, the detection step must be preceded by efficient
disruption of cell walls and membranes. Unfortunately, many pathogens of
the vagina, e.g., Candida albicans, Gardnerella vaginalis, and Group B
streptococci, are extremely difficult to lyse compared with other
microorganisms. Trichomonas lyses easily, but contains potent nucleases
that can easily sabotage diagnostic tests based on detection of nucleic
acids.
Moreover, different methods are currently required to lyse each of these
organisms. As such, the prior art has not provided a general lysis method
that is effective for the simultaneous disruption and release of nucleic
acids for the several pathogens of the vagina. For diagnostic tests
targeted to panels rather than single microorganisms, the use of a
different lysis protocol for each organism would necessitate separate
swabs for each, and the separate processing would drive up the cost of the
test. As a practical matter, a single lysis protocol would be far more
desirable.
One potential biochemical detection method involves the use of nucleic acid
hybridization. The sequence specificity embodied in nucleic acids makes it
possible to differentiate virtually any two species by nucleic acid
hybridization. Standard techniques for detection of specific nucleotide
sequences generally employ nucleic acids that have been purified away from
cellular proteins and other cellular contaminants. The most common method
of purification involves lysing the cells with sodium dodecyl sulfate
(SDS), digesting with proteinase K, and removing residual proteins and
other molecules by extracting with organic solvents such as phenol,
chloroform, and isoamylalcohol.
Endogenous nucleases released during cell solubilization can frustrate
efforts to recover intact nucleic acids, particularly ribonucleic acids
(RNA). While deoxyribonucleses (DNases) are easily inactivated by the
addition of chelating agents to the lysis solution, ribonucleases (RNases)
are far more difficult to eliminate. RNases are ubiquitous, being present
even in the oil found on human hands, and they are practically
indestructible. For example, the standard procedure for preparing
laboratory stocks of pancreatic RNase is to boil a solution of the enzyme
for 15 minutes. The purpose of this treatment is to destroy all traces of
contaminating enzyme activity, since other enzymes cannot survive boiling.
Accordingly, protecting against RNase is a commonly acknowledged aspect of
any standard RNA preparation technique. Sambrook, et al., which is a
compendium of commonly followed laboratory practices, recommends extensive
precautions to avoid RNase contamination in laboratories where RNA work is
conducted. All solutions that will contact RNA are to be prepared using
RNase-free glassware, autoclaved water, and chemicals reserved for work
with RNA that are dispensed exclusively with baked spatulas. Besides
purging laboratory reagents of RNase, RNase inhibitors are typically
included in lysis solutions. These are intended to destroy endogenous
RNases that generally become activated during cell lysis.
From the above descriptions, it is evident that the standard nucleic acid
purification techniques are not practical for the rapid and economical
detection of specific microorganisms outside of a well-equipped
laboratory. Protecting against RNase is cumbersome and costly, and typical
extraction procedures require the handling of caustic solvents, access to
water baths, fume hoods, and centrifuges, and even the storage and
disposal of hazardous wastes. The direct analysis of unfractionated
solubilized microorganisms would avoid the cost and inconvenience of these
purification techniques.
A minimum prerequisite for identifying microorganisms by hybridization is
the release of target nucleic acids from cellular structures that
otherwise would impede entry of the detection probes. Such probes consist
in general of segments of nucleic acid that are complementary to sequences
unique to the target organism. Once the probe has formed a hybrid with the
target, the existence of that hybrid can be ascertained by activating a
signal generating system that is bound to the probe.
Various impediments can block the access of hybridization probes to their
target sequences, the most significant barrier being the cell wall itself.
While the cell walls of many microorganisms can be effectively solubilized
with guanidinium salts or with proteinase K and SDS, these methods do not
effectively release readily hybridizable nucleic acids from many
clinically important microorganisms, e.g., Candida albicans and Gram
positive species. The Gram positive bacteria, which are known to be
difficult to lyse, also do not efficiently yield hybridizable nucleic
acids after treatment with guanidinium salts or proteinase K.
In some instances, unusual mounts of endogenous nucleases have aggravated
the problem of recovering intact nucleic acids. For example, one of the
few groups that has successfully extracted intact DNA from Trichomonas
vaginalis reports that this organism is characterized by a high level of
endogenous nuclease activity, and that its DNA is unusually susceptible to
degradation during isolation. See, Riley, et al., J. Clin. Microbiol.,
30:465-472 (1992).
Moreover, the means available for lysing recalcitrant organisms are often
complex and unwieldy. For example, a common method for the mechanical
lysis of yeast requires the sample to be alternately vortexed with glass
beads and cooled in an ice bath. The cellular extract is recovered by
centrifugation after puncturing the bottom of the tube. Similarly, a
Mini-Beadbeater.TM. has been used for lysing Mycobacterium species, where
cells are ruptured by vigorous shaking with phenol and zirconium beads.
See, Hurley, et al., Journal of Clinical Microbiology, 25:2227-2229
(1987).
The lysis of soil bacteria presents another challenge that has required
drastic measures. Successful methods for their lysis have included
multiple cycles of freeze-thawing, and passage through a French press,
which is a high-pressure shearing device. One recent method for lysing
these bacteria calls for the successive application of sonication,
microwave heating, and thermal shocks. See, Picard, et al., Applied and
Environmental Microbiology, 58:2717-2722 (1992).
Another common approach for lysis of microorganisms has involved enzymes
that attack the cell walls. For example, lyticase has proven effective in
lysing Candida albicans, while achromopeptidase, mutanolysin, or
proteinase K removes cell walls from most Gram positive microorganisms.
See, e.g., Kaneko, et al., Agr. Biol. Chem., 37:2295-2302 (1973); Bollet,
et al., Nucleic Acids Research, 19:1955 (1991); Siegel, et al., Infection
and Immunity, 31:808-815 (1981). However, the use of enzymes in routine
detection protocols is fraught with disadvantages. Chief among these is
cost, but calibration of stock solutions, lengthy incubation times, the
need for low temperature storage, and limited shelf life also make the use
of enzymes less than desirable for protocols involving rapid detection of
microorganisms.
When the microorganisms to be detected are located in human clinical
samples, additional concerns must be accommodated. For one, the presence
of mucous can cause clinical samples from some sources to be viscous and
unmanageable. A successful lysis procedure must disperse mucous and any
other substances that may accompany the sample. Furthermore, the method of
lysis must be compatible with conventional sampling techniques if they are
to be widely accepted by the medical community. For example, samples from
the vagina are customarily taken with a single cotton or dacron swab.
Therefore, samples available for detection of vaginal pathogens normally
will be limited to whatever material that can be eluted from such a swab.
In view of the foregoing, there exists a need for a simple and rapid method
for releasing intact nucleic acid from both prokaryotic and eukaryotic
microorganisms present in a single, biological sample. Moreover, there
exists a need for a simple, fast and effective biochemical method which
selectively detects the microorganisms associated with vaginitis, i.e.,
Gardnerella vaginalis, Trichomonas vaginalis and Candida albicans. The
present invention remedies these needs by providing such methods.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for releasing intact
nucleic acid from a microorganism, the method comprising: combining a
complex biological sample containing the microorganism with a lysis
solution comprising a low ionic strength buffer and a detergent, the lysis
solution having a pH ranging from about 7.0 to about 12.0; and heating the
combined solution to above about 65.degree. C. for more than about five
minutes to release the nucleic acid from the microorganism in the absence
of mechanical force. Using this method, a number of different cells (e.g.,
a prokaryote and a eukaryote) present in a single, biological sample can
be effectively lysed without resorting to the use of enzymes, organic
solvents, glass beads, or bulky machinery (e.g., a French press).
The present invention also provides a method and kit for selectively
detecting a prokaryotic microorganism and a eukaryotic microorganism in a
single, complex biological sample, the method comprising: (a) lysing the
cells of the prokaryotic microorganism and the eukaryotic microorganism by
combining the sample with a lysis solution, thereby releasing nucleic acid
from the microorganisms; (b) contacting the nucleic acid released from the
microorganisms, under hybridizing conditions, with an oligonucleotide
capture probe that selectively hybridizes to the nucleic acid of the
prokaryotic microorganism and an oligonucleotide capture probe that
selectively hybridizes to the nucleic acid of the eukaryotic microorganism
to form a prokaryotic microorganism-capture probe hybridization complex
and a eukaryotic microorganism-capture probe hybridization complex,
respectively; and (c) detecting the hybridization complexes as an
indication of the presence of the prokaryotic microorganism and the
eukaryotic microorganism in the sample.
Moreover, in another aspect of the present invention, a method and kit are
provided for selectively detecting a Group I microorganism selected from
the group consisting of gram positive bacteria, and at least one other
Group II microorganism selected from the group consisting of yeasts,
protozoa, mycoplasmas and gram negative bacteria in a single, complex
biological sample, the method comprising: (a) lysing the cells of a Group
I and a Group II microorganisms by combining the sample with a lysis
solution, thereby intact nucleic acid from the microorganisms; (b)
contacting the nucleic acid released from the microorganisms, under
hybridizing conditions, with an oligonucleotide capture probe that
selectively hybridizes to the nucleic acid of the Group I microorganism
and an oligonucleotide capture probe that selectively hybridizes to the
nucleic acid of the Group II microorganism to form a Group I
microorganism-capture probe hybridization complex and a Group II
microorganism-capture probe hybridization complex, respectively; and (c)
detecting the hybridization complexes as an indication of the presence of
the Group I microorganism and the Group II microorganism in the sample.
Using the methods of the present invention, the following exemplary
organisms can be selectively detected in a single, biological sample:
Gardnerella vaginalis, Trichomonas vaginalis, Candida species (e.g., C.
albicans, C. glabrata, C. kefyr, C. krusei, C. parapsilosis and C.
tropicalis), Group B Streptococci, Prevotella bivia, Ureaplasma
urealyticum, Mobiluncus species, Mycoplasma species, Neisseria gonorrhea,
Chlamydia species and Enterobacteriaceae.
In a further aspect, the present invention provides a method for
determining whether a patient is afflicted with bacterial vaginosis (BV)
that is fast, accurate, and does not require an individual skilled in
identifying clue cells, evaluating wet mounts or the like to assess the
results. The method comprising: (a) determining the pH of a vaginal sample
obtained from the patient; (b) detecting the Gardnerella vaginalis (Gv)
cell level in the vaginal sample in a time period of about 6 hours or
less; and (c) determining that the patient is BV-positive if the pH value
of the vaginal sample is greater than about 4.5 and the Gv cell level of
the vaginal sample is greater than or equal to a critical Gv cell number.
The present invention also provides pharmaceutical and diagnostic kits for
use in the methods of the present invention. For example, the present
invention provides a diagnostic kit for selectively detecting a
prokaryotic microorganism and a eukaryotic microorganism in a single,
complex biological sample, the kit comprising: (a) a dipstick comprising a
nonporous solid support having attached thereto at least two capture
oligonucleotide-coated beads, wherein the first bead selectively
hybridizes to the nucleic acid of a prokaryotic microorganism and the
second bead selectively hybridizes to the nucleic acid of a eukaryotic
microorganism to form a prokaryotic microorganism-capture probe
hybridization complex and a eukaryotic microorganism-capture probe
hybridization complex, respectively; and (b) a container including at
least two signal oligonucleotides, wherein the first signal
oligonucleotide hybridizes to the prokaryotic microorganism and the second
signal oligonucleotide hybridizes to the eukaryotic microorganism.
Additionally, the present invention a diagnostic kit for determining
whether a patient is afflicted with bacterial vaginosis (BV), the kit
comprising: (a) a first indicator capable of indicating a pH greater than
about 4.5; and (b) a second indicator capable of indicating a Gv cell
level greater than or equal to a critical Gv cell number.
Other advantages, objects, features and embodiments of the present
invention will become apparent from the detailed description which
follows.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENT
The present invention provides a method for releasing intact nucleic acid
from a microorganism, the method comprising: combining a complex
biological sample containing the microorganism with a lysis solution
comprising a low ionic strength buffer and a detergent, the lysis solution
having a pH ranging from about 7.0 to about 12.0; and heating the combined
solution to above about 65.degree. C. for more than about five minutes to
release the nucleic acid from the microorganism, wherein the lysis
solution is capable of releasing intact nucleic acid from the
microorganism in the absence of mechanical force.
As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single-stranded or double-stranded form
and, unless otherwise limited, encompasses known analogs of natural
nucleotides which can function in a similar manner as naturally occurring
nucleotides. As used herein, the term "intact" nucleic acid refers to
hybridizable nucleic acid, i.e., nucleic acid of a sufficient length such
that it is capable of hybridizing to an oligonucleotide probe. The term
"complex biological sample" is used herein to refer to a biological
mixture, e.g., vaginal fluid, of nucleic acid (RNA and/or DNA) and
non-nucleic acid. Such a complex biological mixture includes a wide range
of eukaryotic and prokaryotic cells.
Moreover, as used herein, the term "microorganism" refers to both
prokaryotic and eukaryotic microorganisms. The significant differences
between eukaryotic and prokaryotic cells would lead one to believe that a
simple, universal method for releasing intact nucleic acid from a
microorganism would not work well for both kinds of cell, especially the
difficult-to-lyse yeast and the gram-positive bacteria. It has been
discovered, however, that the above lysis method works well for releasing
intact nucleic acid from both eukaryotic and prokaryotic microorganisms,
including, for example, gram-positive bacteria and yeast.
Eukaryotic cells are found in all vertebrates, protozoa, and fungi, while
bacteria exhibit the more primitive prokaryotic cell type. Both eukaryotic
and prokaryotic cells are surrounded by a lipid bilayer that selectively
regulates which molecules may enter or leave the cell. The lipid bilayer
can be ruptured or solubilized by a variety of means, such as suspending
the cells in hypotonic solutions, or treating them with organic solvents,
although such means do not necessarily inactivate nucleases.
In addition to the lipid bilayer, bacteria and some types of primitive
eukaryotic cells are encased by a rigid cell wall that surrounds the
entire cell, including the plasma membrane itself. In bacteria, this tough
protective coat is composed of a carbohydrate matrix cross-linked by short
polypeptide units. (See, Raven and Johnson, Biology, p. 87, Times
Mirror/Mosby College Publishing, 2nd ed., 1989.) No eukaryotes possess
cell walls with a chemical composition of this kind. The most common
methods for lysing bacteria without organic solvents involve treating the
bacteria with lytic enzymes. Lysozyme and mutanolysin are commonly used to
lyse gram-negative and gram-positive bacteria, respectively, but these
enzymes are totally ineffective in lysing eukaryotic cells.
Yeast, which is a type of fungus, also possesses cell walls, but these
differ in composition from those of bacterial cell walls. Cell walls of
yeast rely primarily on .beta.-1,3-glucans for their rigidity. Yeast cell
walls are often stabilized by disulfide bonds that can be disrupted with
mild reducing agents such as .beta.-mercaptoethanol. A number of enzymes,
including lyticase, chitinase, and Novozym.TM., are effective in lysing
some strains of yeast. The lyric activity of lyticase is attributable to
both a .beta.-1,3,-glucanase and a protease and lyses yeast only in the
presence of a reducing agent. (See, Scott and Schekman, J. Bacteriol.
142:414-423 (1980), for a description of lyticase). Novozym.TM., sold by
Novo BioLabs, includes glucanase, proteinase, and chitinase activities. In
experiments performed by Applicants, lyticase, but not Novozym.TM. or
chitinase, was effective in lysing Candida albicans.
Bacteria can be differentiated to some extent according to the composition
of their cell walls. Bacteria are commonly classified according to whether
or not they take up color during a procedure known as the Gram stain.
Cells that incorporate the stain, known as "gram-positive" bacteria, have
a single, thick cell wall that retains the stain and results in their
appearing purple under the microscope. Gram-negative bacteria have evolved
thinner and more complex cell walls that do not retain the stain.
Gram-positive and gram-negative bacteria often differ in their
susceptibility to different kinds of antibiotics as well as in their
susceptibility to various lysis protocols.
Gram-positive bacteria have proven to be exceptionally difficult to lyse
compared with gram-negative bacteria. For example, gram-positive bacteria
are resistant to the inexpensive egg white lysozyme commonly used to lyse
gram-negative microbes (Siegel, et al., Infec. and Immun. 31:808-815
(1981)). As previously discussed, investigators have often resorted to the
use of expensive enzymes to lyse gram-positive bacteria.
Of particular interest is the simultaneous lysis of several pathogenic
microorganisms that infect the human vagina. These microorganisms include,
but are not limited to, Gardnerella vaginalis, Prevotella bivia,
Trichomonas vaginalis, Candida albicans, and several species of Group B
streptococci. Of these, several are difficult to lyse by conventional
means. Trichomonas vaginalis presents a problem because of its reportedly
high endogenous level of nucleases. Candida albicans and the Group B
streptococci are problematic because of their relatively impervious cell
walls. Furthermore, the rapid and economical non-enzymatic lysis of both
eukaryotic and prokaryotic organisms in the same reaction mix is a
challenge not met by any method in the existing art.
As such, a new approach has been discovered that is effective for lysing a
number of different kinds of cells without resorting to the use of
enzymes, organic solvents, glass beads, or bulky machinery. As previously
mentioned, the lysis method of the present invention consists of combining
a complex biological sample containing the microorganism to be lysed with
a lysis solution comprising a low ionic strength buffer and a detergent,
the lysis solution having a pH ranging from about 7.0 to about 12.0; and
heating the combined solution to above about 65.degree. C. for more than
about five minutes to release the nucleic acid from the microorganism,
wherein the lysis solution is capable of releasing intact nucleic acid
from the microorganism in the absence of mechanical force.
In this lysis method, the lysis solution contains a buffer having an ionic
strength ranging from about 15 mM to about 150 mM. Suitable buffers which
can be used for maintaining the pH of the lysis solution include, but are
not limited to, the following: brucine tetrahydrate,
4-(2-hydroxyethyl)-1-piperazinepropane sulfonic acid ("EPPS"),
tris(hydroxymethyl)aminomethane ("TRIS"),
N-tris(hydroxymethyl)methylglycine ("TRICINE"), glycinamide,
N,N-bis(2-hydroxyethyl)glycine ("BICINE"),
N-tris(hydroxymethyl)methyl-2-aminopropane sulfonic acid ("TAPS"),
N-glycyl-glycine, histidine, boric acid, pyrophosphoric acid,
ethanolamine, glycine, trimethylamine,
cyclopentanetetra-1,2,3,4-carboxylic acid, carbonic acid,
3-cyclohexylamino-1-propanesulfonic acid ("CAPS"), EDTA, methylamine,
dimethylamine, ethylmine, triethylamine, diethylamine, ascorbic acid, and
phosphoric acid.
Detergents suitable for use in the lysis method of the present invention
include, but are not limited to, the following: anionic detergents,
cationic detergents, zwitterionic detergents and non-ionic detergents.
Anionic detergents include, but are not limited to, the sodium salts of
caprylic acid, cholic acid, 1-decanesulfonic acid, deoxycholic acid,
glycocholic acid, glycodeoxycholic acid, lauryl sulfate ("SDS"),
N-lauroylsarcosine, taurocholic acid, taurodeoxycholic acid. Cationic
detergents include, but not limited to, cetylpyridinium chloride,
dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and
tetradecyltrimethylammonium bromide. Zwitterionic detergents include, but
are not limited to, CHAPS and CHAPSO. Non-ionic detergent including, but
not limited to, n-decyl .beta.-D-glucopyranoside, digitonin, n-docedyl
.beta.-D-glucopyranoside, n-dodecyl .beta.-D-maltoside, n-heptyl
.beta.-D-glucopyranoside, n-octyl .beta.-D-glucopyranoside, n-octyl
.alpha.-D-glucopyranoside, nonidet P-40, n-nonyl .beta.-D-glucopyranoside,
and Triton X-100.
Additionally, the lysis solution can include a chelating agent (e.g., EDTA)
and/or a preservative (e.g., ProClin.RTM.). In contrast to methods in the
prior art, no enzymes or ribonuclease inhibitors are required in the lysis
solution of the present invention. As such, a presently preferred
formulation of the lysis solution used in the disclosed method for
releasing intact nucleic acid from a microorganism is as follows: 91 mM
N-tris(hydroxy-methyl)aminomethane; 0.5% sodium dodecyl sulfate; 5.0%
N-lauroyl-sarcosine (optional); 10 mM EDTA, and 0.1% ProClin.RTM. 150.
The pH optima of the lysis solution will depend upon which microorganism(s)
is being lysed. In the present format, the pH optima has been determined
for a number of microorganisms of interest, including the bacteria
Gardnerella vaginalis, Prevotella bivia, and Group B streptococci; the
protozoan Trichomonas vaginalis; and the yeast Candida albicans. For
Candida albicans, the optimal pH ranges from a pH of about 10 to about
11.5, but substantial lysis was observed across the entire pH range from
about 6.0 to about 11.5. Likewise, Trichomonas vaginalis lysed well across
the this entire range of pHs, with slightly better results at a pH above
about 8.5. Gardnerella vaginalis lysed well across the entire range, with
a slight decrease in assay signal at pHs above 9.5. The optimal pH for
lysing Group B streptococci was a pH of about 6.0 to abut 8.0, but a
substantial amount of lysis was seen at pHs ranging from about 7.0 to
about 12.0.
It will be readily apparent to those of skill in the art that the pH optima
for other microorganisms can readily be determined. For example, the lysis
conditions for Group B streptococci were optimized using the following
procedure. As a "gold standard" for comparison with the test samples set
forth below, suspensions containing known numbers of freshly-grown
bacteria were lysed with a solution containing about 1 mg/Ml mutanolysin,
2 mg/mL achromopeptidase, 2 mg/mL lysozyme, 2 mg/mL lipase-PN, and 10
mg/mL 20-T Zymolase. After incubating for about five minutes at 37.degree.
C., proteinase K and SDS were added to final concentrations of about 1
mg/ml, and 1.5%, respectively. This mixture was incubated for an
additional five minutes at 60.degree. C. This treatment was designed to
ensure complete lysis by inclusion of all of the enzymes shown previously
to effect lysis of Group B streptococci.
For the test samples, known numbers of freshly-gown bacteria were placed in
a number of test vials and the following lysis solution was added to each
sample: 91 mM N-tris(hydroxy-methyl)aminomethane; 0.5% sodium dodecyl
sulfate; 5.0% N-lauroyl-sarcosine (optional); 10 mM EDTA, and 0.1%
ProClin.RTM. 150. Holding all other factors the same, the pH of the lysis
solution was varied over a pH ranging from about 5.0 to about 12.0.
Similarly, holding all factors the same, the temperature of the lysis
solution was varied over a wide range. The optimal lysis conditions for
Group B streptococci were assessed by comparing the amount of ribosomal
RNA detected in each test sample with the amount of ribosomal RNA detected
in the gold standard lysis mixture. In doing so, it was determined that
Group B streptococci became completely lysed at a temperature of about
85.degree. C., but substantial lysis was seen at temperatures exceeding
65.degree. C. The optimal pH for lysing Group B streptococci ranges from a
pH of about 6.0 to about 8.0, but a substantial amount of lysis was seen
at pH's ranging from about 7.0 to about 12.0. It will be readily apparent
to those of skill that the pH optima for any microorganism can be
determined using a procedure similar to that used for Group B
streptococci.
In a presently preferred embodiment of the lysis method, the combined
solution (i.e., the lysis solution and target microorganism) is heated to
a temperature above about 65.degree. C. for a period of about five to
about ten minutes. More preferably, the combined solution is heated to a
temperature ranging from about 75.degree. C. to about 95.degree. C. Even
more preferably, the combined solution is heated to a temperature of about
85.degree. C. If the lysis temperature exceeds 95.degree. C., little or no
nucleic acid can be detected in the subsequent assays. As such, in
contrast to previously used lysis methods, the use of enzymes, organic
solvents, glass beads, or bulky machinery are not required in the lysis
methods of the present invention.
As a result of this ability to lysis multiple microorganisms in a single,
complex biological sample, assays of any combination of microorganisms
discussed above can be conducted in the same reaction mixture, thereby
making it possible to devise diagnostic assays for different
microorganisms that may be present in the same complex biological sample,
e.g., the same patient sample. This approach is useful for devising assays
for pathogens all of which are associated with the same clinical symptoms.
For example, the vaginitis organisms Gardnerella vaginalis, Candida
albicans, and Trichomonas vaginalis can all be in a single sample if the
microorganisms are first lysed to release their nucleic acid using the
lysis method of the present invention. As such, a single sample from
pregnant women can be assessed for the presence of multiple organisms
which are known to cause premature birth. For example, a prenatal assay
panel can include Trichomonas vaginalis, Prevotella bivia, Gardnerella
vaginalis, and Group B streptococci or, a subset thereof. As this lysis
method works on such a great variety of microorganisms, a wide variety of
combinations of organisms can be assayed by analyzing single patient
samples from any part of the body.
As such, in another aspect of the present invention, a method and kit are
provided for selectively detecting a prokaryotic microorganism and a
eukaryotic microorganism in a single, complex biological sample, the
method comprising: (a) lysing the cells of the prokaryotic microorganism
and the eukaryotic microorganism by combining the sample with a lysis
solution, thereby releasing nucleic acid from the microorganisms; (b)
contacting the nucleic acid released from the microorganisms, under
hybridizing conditions, with an oligonucleotide capture probe that
selectively hybridizes to the nucleic acid of the prokaryotic
microorganism and an oligonucleotide capture probe that selectively
hybridizes to the nucleic acid of the eukaryotic microorganism to form a
prokaryotic microorganism-capture probe hybridization complex and a
eukaryotic microorganism-capture probe hybridization complex,
respectively; and (c) detecting the hybridization complexes as an
indication of the presence of the prokaryotic microorganism and the
eukaryotic microorganism in the sample.
In accordance with this method and kit of the present invention, a | | |
